Differential regulation by agonist and phorbol ester of cloned m1 and m2 muscarinic acetylcholine receptors in mouse Y1 adrenal cells and in Y1 cells deficient in cAMP-dependent protein kinase

Biochemistry. 1990 Sep 11;29(36):8475-83. doi: 10.1021/bi00488a039.

Abstract

Cloned muscarinic acetylcholine m1 and m2 receptors were expressed in stably transfected mouse Y1 adrenal cells and in a variant Y1 line, Kin-8, which is deficient in cAMP-dependent protein kinase activity (PKA-). m1 and m2 receptors were rapidly internalized following exposure of transfected PKA+ or PKA- cells to the muscarinic agonist carbachol. Thus, agonist-dependent internalization of m1 and m2 did not require PKA activity. A differential effect of PKA on regulation by agonist of the m2 receptor, but not the m1 receptor, was unmasked in PKA- cells. The m2 receptor was more sensitive to agonist-dependent internalization, and its rate of internalization was faster in PKA- cells than it was in PKA+ cells. Treatment of PKA+ cells with 8-(4-chlorophenylthio)-cAMP or forskolin did not result in internalization of either m1 or m2 receptors and did not alter the extent of agonist-dependent internalization of m2. These data indicate that the basal activity of PKA may modulate the agonist-dependent internalization of the m2 receptor, but not the m1 receptor. The internalization of the m1 and m2 receptors in both PKA+ and PKA- cells was accompanied by desensitization of functional responses. Exposure of PKA+ cells to 10(-7) M phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, resulted in a 30 +/- 9% decrease in the number of m1 receptors on the cell surface. However, treatment of PKA- cells expressing the m1 receptor did not result in internalization, suggesting that PKA was required for some aspect of PMA-dependent internalization.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenocarcinoma / pathology
  • Adrenal Gland Neoplasms / pathology
  • Animals
  • Colforsin / pharmacology
  • Concanavalin A / pharmacology
  • Cyclic AMP / analogs & derivatives
  • Cyclic AMP / metabolism
  • Cyclic AMP / pharmacology
  • Endocytosis / drug effects
  • Mice
  • N-Methylscopolamine
  • Phorbol Esters / pharmacology
  • Pilocarpine / pharmacology
  • Protein Kinases / deficiency
  • Protein Kinases / physiology*
  • Quinuclidinyl Benzilate / metabolism
  • Receptors, Muscarinic / classification
  • Receptors, Muscarinic / drug effects*
  • Receptors, Muscarinic / metabolism
  • Recombinant Fusion Proteins
  • Scopolamine Derivatives / metabolism
  • Second Messenger Systems / drug effects*
  • Thionucleotides / pharmacology
  • Tumor Cells, Cultured / drug effects

Substances

  • Phorbol Esters
  • Receptors, Muscarinic
  • Recombinant Fusion Proteins
  • Scopolamine Derivatives
  • Thionucleotides
  • Pilocarpine
  • Concanavalin A
  • Colforsin
  • 8-((4-chlorophenyl)thio)cyclic-3',5'-AMP
  • Quinuclidinyl Benzilate
  • Cyclic AMP
  • Protein Kinases
  • N-Methylscopolamine